Summary and Abstract RNA splicing is the process of removal of intronic sequences from the primary RNA transcript before the final mRNA is generated. Unlike lower eukaryotes, the vast majority of mammalian genes are spliced. Most genes give rise to multiple mRNAs resulting from differential promoters, termination sequences, or the use of alternative exons. Although often depicted as sequential steps, transcription and splicing are now thought to occur simultaneously, however supporting evidence is scarce. More importantly, how alternative splice sites are recognized in the context of co-transcriptional splicing is unknown. Insulin is essential for growth and development in addition to fuel metabolism. There are two variants of the insulin receptor (IR), which differ in the presence of 12-amino acids in the hormone-binding domain. The two variants arise from alternative splicing of exon 11. The IR lacking exon 11 is widely expressed and binds both insulin and IGF-II; the IR containing exon 11 is expressed predominantly in the insulin-sensitive tissues liver, muscle, adipocytes and kidney, and only binds insulin. More importantly, a number of disease states, such as type II diabetes, aging, myotonic dystrophy and cancer, have decreased inclusion of exon 11. This makes the INSR gene a particularly interesting model system for the study of RNA splicing. Based on our extensive preliminary data we are proposing a comprehensive but realistic series of experiments to test two alternative models of co-transcriptional INSR gene splicing. These studies will address key questions concerning the fundamental biological process of co-transcriptional alternative splicing and will integrate cell and molecular biological experiments with physiological studies in mice lacking specific splicing factors in liver. Specific Aim #1: To test for co-transcriptional splicing and the kinetic competition model for alternative exon recognition. We will attempt to catch the spliced RNA still associated with chromatin using the new ChRIP method and will determine whether there is a transcriptional pause near exon 11. To test sufficiency, an artificial pause site will be engineered downstream of exon 11 and transcriptional elongation rates will be modulated genetically and pharmacologically. Specific Aim #2: To determine whether SRp20 or SF2 is required for transcriptional pausing and co- transcriptional splicing of the INSR gene. We will test whether exon 11 requires SRp20 or SF2 for association with chromatin, whether there is either a SRp20 or SF2-dependent transcriptional pause near exon 11, and whether SRp20 and SF2 co-localize at the pause site. We will also test whether elevated levels of hnRNP-A1 in HEK293 cells prevents co-transcriptional splicing via interfering with SF2 binding. Specific Aim #3: To determine whether phosphorylation of SRp20 is required for co-transcriptional splicing of the INSR gene. We will test whether PPP1R10 targets PP1-type phosphatases to exon 11 to dephosphorylate SRp20, preventing its release from chromatin and reducing exon inclusion. We will also test whether PP1 activity is regulated by PKA and insulin and whether PPP1R10 binds to RNA or via CUG-BP1. Specific Aim #4: To create genetic liver-specific knock-outs of SRp20 and SF2. Mice will be created by crossing SRp20flox/flox and SF2flox/flox mice with albumin-cre mice to delete the two splicing factors in hepatocytes. These mice should preferentially express the IR-A isoform. We will determine whether these mice are insulin-resistant using a panel of metabolic tests and we will assess other potential targets for SRp20 and SF2 in the liver using genomic approaches. PUBLIC HEALTH RELEVANCE: Diabetes mellitus is epidemic and expected to double in the next 20 years. An estimated 15% of VA patients have diabetes and >95% of these have Type 2 diabetes. Insulin resistance is even more common, affects an estimated 42-43% of people between ages 60-80, and is a feature of many metabolic syndromes including obesity, aging, hypertension, atherosclerosis, diabetes, myotonic dystrophy, glucocorticoid excess, and polycystic ovary syndrome. Alterations in alternative splicing of the insulin receptor gene is observed in a number of these states and a shift in insulin receptor expression from the insulin sensitive B isoform to the less metabolically active A isoform may contribute to insulin resistance and Type 2 DM. The aging VA patient population is also at risk for neurodegenerative diseases and cancer and these disorders are also associated with defects in RNA splicing. Therefore, a detailed understanding of the mechanisms involved in regulating alternative splicing may have wide applicability to many diseases that plague the VA population.